Multiplex Assays
Multiplexing or multiplex assay refers to the ability to send, receive, detect, separate, differentiate, or analyze multiple signals or streams of information simultaneously, from the same source. Multiplexing of assays include the ability to: conduct multiple tests (e.g., chemical assays, biological assays, immunosorbent assays, immuno-assays, and the like); measure or detect multiple items (e.g., antigen concentration, antibody concentration, analytes such as cytokines, chemokines, inflammatory mediators, and their receptors that are present in biological fluids (e.g. serum, plasma, urine, tear, cell culture supernatants, intact cells or their lysates), functionalized microspheres, eTags, labeled moieties, and the like); or manipulate multiple items (e.g., animal cells, bacteria, viruses, their culture supernatants, enzymes, macromolecules, or their mixtures) individually, at the same time, or within the same sample, or within the same test tube/chamber/well/compartment.
Examples of current multiplex biological assays include DNA microarrays (used, for example, for gene expression profiling or for the detection of nucleotide sequence mutations or deletions); assays in combinatorial chemistry (for the analysis of multiple closely-related chemical compounds); and immunology-based assays (such as the Enzyme-Linked Immunosorbent Assay (ELISA) used to detect and quantify antigens), among others.
Existing multiplex assays are generally limited by the number of analytes they can simultaneously detect or differentiate. In the following text, some characteristics of current multiplex assay systems will be discussed in the context of ELISA systems, which embody many of the characteristics shared by most existing biological multiplex systems. Current multiplexing systems, such as standard ELISA, allow for the quantitation of one analyte per each well of sample. Typically, in these systems, a sample is divided among several wells of a microplate. Currently, 96-well ELISA plates are commonly used and require up to 100 μL of sample per well or up to 9.6 mL of sample per plate. The 384-well and 1536-well ELISA plates are also becoming available. These plates require a smaller volume of sample per well than 96-well plates, however, a relatively large volume of sample is still needed per plate. See, for example, Wu et al. J. Clin. Lab. Anal. 17:241-246 (2003). One drawback of standard multiplex assays is that they require a relatively large amount of sample to measure more than one analyte. Another drawback is that liquid handling becomes a problem for microplate-based assays with a higher number of wells. Usually, sophisticated and expensive liquid handling systems must be used to dispense and wash samples in these plates.
To scale down the amount of sample needed for multiplex assays, multiplex fluorescent bead-based immunoassays have been developed that allow the measurement of several proteins in a single sample well. These systems utilize a combination of several fluorescently-distinguishable microsphere beads, each covalently coupled to an antibody that can specifically capture a particular type of protein within the sample. Normally, a handful of specific capture beads are mixed with a minimum volume of the sample (in the microliter range) in each well of a 96- or higher number-well microplate. Each microsphere bead can act as an independent solid surface to which a specific analyte can be attached and be subsequently detected. See, for example, Kellar et al., Cytometry 45:27-36 (2001). A major drawback of these systems is again the need for a relatively large volume of sample and reagent per microplate for detecting a handful of analytes. Thus, these assays can be costly, and at times unsuitable for those instances where there is only a very small quantity of sample available. A further drawback is the liquid handling problem that arises when using 384- or higher number-well ELISA microplates. Liquid handling and sample evaporation become major problems when dealing with small volumes of samples in an open system. An additional drawback of the current multiplex ELISA systems, which also applies to standard ELISA assays, is the possibility for contamination and cross-contamination of samples since plates are open to the environment.
Yet another limitation of existing multiplex systems is that, due to relatively large microliter-scale volume of sample used per well, each analyte must be assayed with multiple beads of the same type to prevent signal saturation. Similar beads will compete with each other to bind to the same analyte. This situation decreases the sensitivity of the assay because the target analyte present in the sample is distributed over all of the beads specific for that analyte; and each bead will be reporting only a fraction of the analyte concentration. The mean value of the analyte concentration will, therefore, have a large standard error due to variable concentration values reported by each bead.
As a standard operating protocol, microplates in microplate-based assays are vortexed or rocked at room temperature for an extended period of time to allow the analytes to be mixed well with functionalized microbeads in each well. The drawback of such a mixing procedure is that it could lead to sample evaporation, spilling, cross-contamination, and analyte degradation.
A further limitation with existing multiplex systems is that they test samples that come from tissue culture supernatants or biological fluids that contain analytes produced by mixed cell populations. The current multiplexing systems cannot provide direct information about the analyte profiles of individual cells or if they do their level of multiplexing is very limited. Another drawback of the current multiplex systems is that they are unable to detect expression kinetics of different analytes instantaneously as they become expressed or secreted (e.g., when analytes are secreted from a live cell). Currently, in order to study, for example, the kinetics of cytokine expression in cells after a particular treatment, culture supernatants must be collected at various time points after the treatment and be frozen and saved until samples from all time points are gathered. Subsequently, samples are thawed out and applied to multi-well plates for studying cytokine expressions over time. A major drawback of such a procedure is that it fails to properly quantify any analyte that is short lived or is degraded as the result of lengthy experimental procedures, improper laboratory handling, freeze-thawing, and the like.
Consequently, to alleviate the shortcomings of current multiplexing systems, there is a need for devices and methods capable of detecting multiple analytes in a single sample using sub-microliter volumes, thereby reducing the cost and the need for large volume of samples and reagents. There is also a need to automate the liquid handling and the assaying procedures to reduce costly laboratory time and effort required to perform larger-scale assays. There is a further need for assays to be performed in an enclosed environment so that sample evaporation, spilling, contamination, and cross-contamination are eliminated. There is also a need for a system that can detect the analyte profile at a single-cell level. There is a further need for a single-cell system to be capable of capturing analytes as they are secreted from the cell. Moreover, there is a need for a single-cell assay to be capable of sampling analytes at several time intervals so that the kinetics of analyte expression or secretion from the cell can be measured over time.
The devices and methods of the present invention provide the tools to accomplish these objectives by using microfluidic systems.
Microfluidic Systems
Microfluidics refers to a set of technologies that control the flow of nanoliter and picoliter amounts of fluids in miniaturized systems.
It is not generally possible to scale down existing assaying methods and devices and expect them to work in microfluidics applications. One problem encountered is that the behavior of a fluid changes dramatically at the micron scale. For example, capillary action can have an effect on how fluids pass through microscale-diameter tubes. Additionally, other factors, such as heat transfer and mass transfer, can have a different effect in micro-scale systems, as compared to macroscale systems. Another problem associated with the scale down of existing assaying methods is that fluid viscosity dominates over momentum, thus resulting in, among other things, problems with mixing.
The advent of “multilayer soft lithography” (MSL) techniques and multilayer microfluidic systems made by it, however, have solved some of the problems mentioned above in the field of microfluidics. In MSL, multilayer structures are constructed by binding multiple patterned layers of elastomers. In such structures, the fluid flow in microfluidic channels of one layer (the control layer) provides an actuation force necessary to control the fluid flow in another layer (the flow layer). As a result, the active multilayer microfluidic devices produced by MSL techniques can have multiple valves and pumps completely made out of elastomers. Such multilayer microfluidic devices are superior to microelectromechanical structures (MEMS) in that they are not limited by the common problems associated with moving liquid by techniques such as electroosmotic flow or dielectrophoresis . See, for example, Unger et al., Science 288:113-116 (2000).
Existing MEMS microfluidic systems and technologies have found utility in ink-jet print heads, fabrication of DNA microarrays, and DNA analysis technologies, among a myriad of other applications. See, for example, Chen et al., Combinatorial Chemistry & High Throughput Screening 7(1):29-43 (2004). These systems and technologies, however, employ complicated fabrication techniques, and utilize fragile and expensive components. Moreover, many of these systems are only adapted to a single type of experiment, technique, or use.
Accordingly, the present invention eliminates the limitations of current multiplex assays by providing MSL-based microfluidic devices and methods that can replace macroscopic laboratory tools with smaller, automated, and more efficient tools that are adaptable to more than one experiment, technique, or use. The present invention further provides low cost, quantitative, microfluidic assaying methods and devices adaptable to biotechnological, biological, biomedical, pharmaceutical, chemical, or environmental uses, among others. The present invention provides a straightforward solution to the challenge of measuring multiple analytes within each chamber within closed microfluidic devices.